title: Biochemical and biophysical analysis of a chiral PqsD inhibitor revealing tight-binding behavior and enantiomers with contrary thermodynamic signatures.
abstract: Antivirulence strategies addressing bacterial pathogenicity without exhibiting growth inhibition effects represent a novel approach to overcome today's crisis in antibiotic development. In recent studies, we examined various inhibitors of PqsD, an enzyme involved in formation of Pseudomonas aeruginosa cell-to-cell signaling molecules, and observed desired cellular effects for 2-nitrophenyl derivatives. Herein, we investigated the binding characteristics of this interesting compound class using several biochemical and biophysical methods. The inhibitors showed time-dependent activity, tight-binding behavior, and interactions with the catalytic center. Furthermore, isothermal titration calorimetry (ITC) experiments with separated enantiomers revealed contrary thermodynamic signatures showing either enthalpy- or entropy-driven affinity. A combination of site-directed mutagenesis and thermodynamic profiling was used to identify key residues involved in inhibitor binding. This information allowed the proposal of experimentally confirmed docking poses. Although originally designed as transition state analogs, our results suggest an altered position for both enantiomers. Interestingly, the main difference between stereoisomers was found in the orientation of the hydroxyl group at the stereogenic center. The predicted binding modes are in accordance with experimental data and, thus, allow future structure-guided optimization.
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) , Department Drug Design and Optimization, Campus C2.3, 66123 Saarbrücken, Germany.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Storz
authors.forename: Christian
authors.initials: C
authors.lastname: Brengel
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Weidel
authors.forename: Michael
authors.initials: M
authors.lastname: Hoffmann
authors.forename: Klaus
authors.initials: K
authors.lastname: Hollemeyer
authors.forename: Anke
authors.initials: A
authors.lastname: Steinbach
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.forename: Martin
authors.initials: M
authors.lastname: Empting
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Hartmann
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D053038:Quorum Sensing / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins